Method of producing FR901228

ABSTRACT

Depsipeptides and congeners thereof are disclosed having structure (I), wherein m, n, p, q, X, R1, R2 and R3 are as defined herein. These compounds, including FR901228, have activity as, for example, immunosuppressants, as well as for the prevention or treatment of patients suffering or at risk of suffering from inflammatory, autoimmune or immune system-related diseases including graft-versus-host disease and enhancement of graft/tissue survival following transplant. Also provided are methods for inhibiting lymphocyte activation, proliferation, and/or suppression of IL-2 secretion

TECHNICAL FIELD

The present invention relates to a method of producing FR901228 which isuseful as an antibacterial agent and an antitumor agent. Moreparticularly, it relates to a method of producing FR901228 whichcomprises culturing a FR901228 producing strain in a medium added withat least one amino acid selected from the group consisting ofL-arginine, L-histidine, L-cystine and L-cysteine or salt thereof.

BACKGROUND ART

FR901228 is a compound produced by culturing a strain belonging toChromobacterium, e.g., Chromobacterium violaceum WB968 strain (FERMBP-1968) in a nutrient medium, and represented by the following formula(Japanese Patent Publication No. Hei 7 (1995)-64872):

In addition to the fermentation method described above, it is known thatFR901228 can also be prepared by semisynthesis or whole synthesisutilizing techniques known in the art (J. Am. Chem. Soc., 118, 7237-7238(1996)).

FR901228 is known to have a histone deacetylase inhibiting activity(Nakajima, H et al., Experimental Cell Research, 241, 126-133 (1998)),and it has been proposed to expand its utility as an antibacterial agentand an anticancer agent.

However, the fermentation method shows an unsatisfactory productiontiter of FR901228. Accordingly, it has been demanded a discovery of astrain excellent in FR901228 producing ability or development of aproduction method capable of increasing the yield of FR901228.

DISCLOSURE OF INVENTION

The inventors of the present invention have noticed the cultureconditions for the fermentation method. Upon investigation of theaddition of amino acids to a medium, they have found that the additionof specific amino acids represented by L-cysteine unexpectedly increasesthe yield, though valine or threonine considered to be possiblecomponents of FR901228, as well as methionine regarded as effective forproduction of S-containing compounds do not show increase in yield ofFR901228. Thus, the present invention has been achieved.

According to the present invention, provided is a novel fermentationmethod of producing FR901228, more particularly, a method of producingFR901228 which comprises culturing a FR901228 producing strain in amedium added with at least one amino acid selected from the groupconsisting of L-arginine, L-histidine, L-cystine and L-cysteine or saltthereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph for illustrating a comparison of yields of FR901228 inaccordance with the addition of various cysteine-related compounds;

FIG. 2 is a graph for illustrating a result of comparative examinationon an optimum concentration of L-cysteine added in a medium;

FIG. 3 is a graph for illustrating a result of powder X-ray diffractionanalysis of type A crystals (crystals obtained from ethanol/water);

FIG. 4 is a graph for illustrating a result of powder X-ray diffractionanalysis of type A crystals (crystals obtained from acetone/water); and

FIG. 5 is a graph for illustrating a result of powder X-ray diffractionanalysis of type B crystals.

BEST MODE FOR CARRYING OUT THE INVENTION

As the FR901228 producing strian, used are strains belonging to theabove-described Chromobacterium, more specifically, Chromobacteriumviolaceum WB968 strain (FERM BP-1968: deposited with NIBH on 20 Jul.1988 under Budapest Treaty). Any other producing strains than the abovemay be utilized as long as they can produce FR901228. They may bespontaneous mutation strains, or artificial mutation strains obtained byX-ray irradiation, UV irradiation, or treatment with various chemicalsubstances such as N-methyl-N′-nitro-N-nitrosoguanidine, 2-aminopurineand the like.

The amino acid used in the method of the present invention is selectedfrom the group consisting of L-arginine, L-histidine, L-cystine andL-cysteine, which may be singly or in combination of two or more kindsthereof. Further, the amino acid may be in the form of its acid additionsalt with an acid such as hydrochloric acid, phosphoric acid, aceticacid, citric acid, succinic acid, lactic acid, tartaric acid, fumaricacid and maleic acid.

An amount of the amino acid added to the medium may vary a littledepending on the kind of the amino acid. The amino acid however may begenerally added to be its concentration of 2 mM or more, preferably 2 to30 mM in total with respect to the medium in a volume of 1 liter. Withsuch addition amount, FR901228 is produced in an increased yield.

In case two or more amino acids are mixed, those amino acids may beoptionally mixed without any particular limitation to the combinationand ratio thereof. However, according to the inventors' experiments, itis confirmed that the addition of L-cysteine shows remarkable increasein the yield of FR901228 as compared with other amino acids that areadded individually. Accordingly, where the amino acid mixture is used,it is considered that the yield will be more increased by mixingL-cysteine as one of components of the mixture in a greater mixingratio.

FR901228 is efficiently produced by culturing the FR901228 producingstrain in a nutrient medium containing at least the above-describedamino acid and a carbon source and a nitrogen source that can beutilized by the FR901228 producing strain under an aerobic condition.

Examples of the carbon source include glucose, galactose, starch,fructose, dextrin, glycerin, maltose, arabinose, mannose and the like.Examples of the nitrogen source include inorganic or organic nitrogencompounds such as bouillon, yeast extract, peptone, gluten meal,cottonseed flour, soy flour, corn steep liquor, dried yeast, ammoniumsalts (e.g., ammonium nitrate, ammonium sulfate and ammonium phosphate),or urea and the like.

It is preferred to combine a single carbon source and a single nitrogensource both of high purity, but those of low purity each containing asmall amount of a growth factor and a considerable amount of aninorganic nutrient may be used. These sources may properly be useddepending on needs.

The above-described medium may contain an inorganic salt such as analkali metal carbonate (e.g., sodium carbonate, potassium carbonate andthe like), an alkali metal phosphate; a magnesium salt (e.g., magnesiumsulfate and the like); a copper salt (e.g., cupric nitrate) and a cobaltsalt (e.g., cobalt acetate); and liquid paraffin, fatty oil, vegetableoil, mineral oil as well as an antifoaming agent such as silicon and thelike, if required.

Preferably, the culture is conducted under deep aerobic condition in alarge scale and under shaking or surface culture condition in a smallscale. Where the strain is cultured in a large tank, it is preferred touse a preculture of the strain as a seed culture. For example, thepreculture may be prepared by inoculating spores or hyphae of the strainin a relatively small amount of a medium and culturing the inoculatedmedium. In such a case, the medium used for preparing the preculture maysubstantially be the same as or different from a medium used forproduction of FR901228 performed later.

During the culture, stirring and aeration may be carried out by variousmethods known in the art. For example, the stirring may be performed byusing various stirrers such as a propeller or a pump equipped with aculture apparatus, or by shaking or rotating the apparatus itself.Alternatively, the stirring and aeration may be performed simultaneouslyby passing sterilized air in the culture.

The culture may be generally carried out at a temperature in the rangeof about 10 to 40° C., preferably about 25 to 35° C., for a period ofabout 15 to 50 hours. The condition may appropriately be varieddepending on various factors such as culture scale and the like.

FR901228 obtained by the above-described culture may be isolated andpurified by conventional methods, e.g., solvent extraction,concentration under reduced pressure, filtration, pH adjustment,adsorption treatment using an inorganic adsorbent, an adsorption resinor the like, crystallization and the like, or by a combination thereof.

For example, FR901228 is a substance produced in the inside of thestrain cells and should be isolated from the strain cells. Accordingly,for easy isolation of FR 901228, pH is adjusted to 1 to 4, preferably1.5 to 2 by suitably adding an inorganic acid such as sulfuric acid,hydrochloric acid, phosphoric acid, nitric acid or the like; or anorganic acid such as citric acid, acetic acid, malic acid, lactic acidor the like to the culture solution.

Examples of the inorganic adsorbent used for the adsorption treatmentinclude silica gel, porous ceramic and the like. As the adsorptionresin, DIAION HP10, DIAION HP20, DIAION HP21, DIAION HP40, DIAION HP50and the like (trademark, manufactured by Mitsubishi ChemicalCorporation) may be used.

EXAMPLES

Hereinafter, the production method of the present invention will beexplained in detail by way of examples, but the invention is not limitedthereto.

Culture:

A medium (20 ml) containing glucose (1%) and bouillon (2%) was put in a100 ml Erlenmeyer flask, and sterilized at 121° C. for 20 minutes.

A loopful of slant culture of Chromobacterium violaceum WB 968 wasinoculated on the medium and cultured at 30° C. for 24 hours on a rotaryshaker.

Another medium (20 ml) containing glucose (1% w/v), bouillon (2% w/v),monopotassium phosphate (1.1% w/v), disodium phosphate dodecahydrate(0.72% w/v), ammonium sulfate (0.1% w/v) and magnesium sulfateheptahydrate (0.006% w/v) was put in a 100 ml Erlenmeyer flask andsterilized at 120° C. for 20 minutes. To this medium, a part of theculture obtained above (400 μl) was inoculated. Further, glucosesterilized at 120° C. for 20 minutes (40%, 1 ml) and filter-sterilized(with a membrane filter having pores of 0.45 μm or less) various aminoacid solutions (200 mmol/l) or sterilized water containing no amino acid(500 μl) were added to the medium. Then the culture was conducted at26.5° C. for 48 hours in a rotary shaking culture apparatus.

Example 1

As the amino acid added to the medium in the above-described culturemethod, L-amino acids of 5 mM/L each described in Table 1 below wereused and the influence of the amino acids upon the FR901228 productionwas examined.

An amount of FR901228 in the culture solution was measured by highperformance liquid chromatography (column: Mightysil RP-18 GP (particlesize: 3 μm), manufactured by KANTO KAGAKU, 3.0 mm I.D.×150 mm; columntemperature: kept constant around 35° C.; detection wavelength: 210 nm;flow rate: 0.3 ml/min; injection amount: 2 μl; mobile phase:THF/acetonitrile/water/phosphoric acid (570:380:50:1)). The culturesolution was centrifuged and the resulting strain was dried and theweight was measured. Thus an amount of dried strain was obtained and itsamount with the respect to the culture solution was calculated.

TABLE 1 Amount of Amount of Amount of FR901228 FR901228 dried strain(×10³)/ (μg/ml) (mg/ml) amount of dried starin Control 151 17.8 8.5(without amino acid) Arg 208 20.9 10.0 His 204 21.4 9.5 Cys 224 18.312.2 Val 128 17.0 7.5 Thr 150 17.0 8.9 Gly 131 15.8 8.2 Met 152 17.8 8.5

As shown in Table 1, a yield of FR901228 was increased through theaddition of arginine (Arg), histidine (His) or cysteine (Cys). Arginineand histidine also increased the amount of dried starin together withthe addition, but cysteine did not show change in the amount of driedstrain as compared with the control. Accordingly, the yield per driedstrain amount was remarkably high when the cysteine was added.

Unexpectedly, valine (Val), threonine (Thr) and glycine (Gly) that werepossible components of FR901228, as well as methionine (Met) which wasan amino acid containing a sulfur atom did not show increase in yield ofFR901228.

Example 2

In the same manner as in Example 1, various cysteine-related compoundswere used to examine the influence of them on the production ofFR901228. L-cystine (L-Cys-Cys) was used in a ½ amount.

As a result, L-cysteine (L-Cys), its hydrochloride (Cys.HCl) andL-cystine showed increase in yield of FR901228 as shown in FIG. 1.

Although the cysteine moiety in FR901228 was in the D form, the yield ofFR901228 was not increased by addition of D-cysteine (D-Cys). Further,the yield was not increased though dithiothreitol (DTT), reducedglutathione (GSH) or acetylated cysteine compound, i.e.,N-acetyl-L-cysteine (Ac-L-Cys), each of which was a compound containinga SH group, was added respectively.

Example 3

In the same manner as in Example 1, an optimum concentration ofL-cysteine added in the medium was examined and the results shown inFIG. 2 were obtained. Amino acid solution of 500 mmol/L was used.

From the results, it was found that the concentration of L-cysteine inthe medium was not directly related to the yield of FR901228.

Example 4

From the control sample without amino acid prepared in Example 1,FR901228 was isolated and purified by the following method.

A culture solution (2190 ml) was prepared in a scale of 100 timesgreater than that described in the section of Culture. After theculture, pH was adjusted to 2.0 with 1N sulfuric acid and the culturesolution was filtrated. The strain was washed with water and the washingwater was combined with the filtrate obtained in the previous step. Thecombined solution (5000 ml) was introduced in a column containing anadsorption resin DIAION HP20 (trademark, manufactured by MitsubishiChemical Corporation, 54 ml). The column was washed with water (100 ml)and 25% aqueous acetone (100 ml) and then eluted with 65% aqueousacetone.

The eluate was diluted with water to obtain water content of 70% orhigher. The diluted solution (720 ml) was introduced in a columncontaining DIAION HP20SS (trademark, manufactured by Mitsubishi ChemicalCorporation, 40 ml), washed with 40% aqueous acetone (160 ml) and theneluted with 47% aqueous acetone.

The eluate (120 ml) was diluted with water to obtain water content of70% or higher. Thus obtained solution (200 ml) was introduced in acolumn of DIAION HP20 (9 ml), washed with 20% aqueous acetone (18 ml)and then eluted with acetone (50 ml). The eluate was concentrated todryness under reduced pressure and the resulting residue was dissolvedin ethyl acetate (3 ml). The solution was introduced in a column ofsilica gel (silica gel 60, 70-230 mesh, 60 ml), which is previouslyfilled with n-hexane:ethyl acetate (1:1 v/v). The column was developedwith n-hexane:ethyl acetate (1:1 v/v, 180 ml) and n-hexane:ethyl acetate(1:2 v/v). Fractions containing FR901228 were combined and concentratedunder reduced pressure. The residue was dissolved in acetone (20 ml),which was then added with methanol and concentrated under reducedpressure to obtain FR901228 (250 mg).

Purification-1:

FR901228 (150 mg) was dissolved in 85% ethanol in a concentration of 94mg/ml. To this solution, water in an amount of 0.8 times greater thanthat of the solution was added over about 10 minutes (concentration of52 mg/ml), and then water in an amount of 4.2 times greater was addedover about 3 hours (final concentration of about 15 mg/ml). After thetotal amount of water was added, the precipitate was collected byfiltration to give type A crystals of FR901228 (100 mg).

The crystals obtained from ethanol/water were measured by using a powderX-ray diffraction apparatus, Philips MPD 1880 under the followingconditions: voltage 40 kv; current 30 mA; Gonio meter PW1775; scanningmode continuous; rate of 0.10 deg/s; distance 0.02 deg; sampling time0.20 s; DS 1°; RS 0.2 mm; and SS 1°. FIG. 3 shows the results.

Purification-2:

FR901228 (150 mg) was dissolved in 85% aqueous acetone in aconcentration of 94 mg/ml. To the solution, water in an amount of 0.8times greater than that of the solution was added over about 10 minutes(concentration of 52 mg/ml), and then water in an amount of 4.2 timesgreater was added over about 3 hours (final concentration of about 15mg/ml). After the total amount of water was added, the precipitate wascollected by filtration to give type A crystals of FR901228 (120 mg).

The crystals obtained from acetone/water were subjected to the powderX-ray diffraction in the same manner as in Purification-1. FIG. 4 showsthe results.

Purification-3:

FR901228 (510 mg) was dissolved in acetone in a concentration of about11 mg/ml. The solution containing FR901228 was cooled to 5° C. Aftercooling, n-hexane in an amount equivalent to that of the solution wasadded over about 20 minutes (concentration of about 5.6 mg/ml) and thenn-hexane was added in an amount of 8 times greater than that of thesolution over about 70 minutes (final concentration of about 1.2 mg/ml)while maintaining the temperature at 10° C. or lower. After the totalamount of n-hexane was added, the precipitate was collected byfiltration to give type B crystals of FR901228 (465 mg).

FIG. 5 shows the results of the powder X-ray diffraction of the type Bcrystals (diffraction conditions were the same as those inPurification-1).

According to the present invention, provided is a method of producingFR901228 which comprises culturing a FR901228 producing strain in amedium added with at least one amino acid selected from the groupconsisting of L-arginine, L-histidine, L-cystine and L-cysteine or saltthereof.

According to this method, FR901228 can be produced more efficiently thanconventional methods using no amino acid. Therefore, the utilization ofFR901228 having high industrial utility value can be further promoted.

1. A method of producing FR901228 which comprises culturing a producingstrain for FR901228 represented by the following formula:

in a medium added with at least one amino acid selected from the groupconsisting of L-arginine, L-histidine, L-cystine and L-cysteine or saltthereon.
 2. A method according to claim 1, wherein the amino acid isL-cysteine.
 3. A method according to claim 1, wherein the amino acid isadded in a concentration of 2 to 30 mM with respect to a medium in avolume of 1 liter.
 4. A method according to claim 2, wherein the aminoacid is added in a concentration of 2 to 30 mM with respect to a mediumin a volume of 1 liter.